【作者】 刘华；

【导师】 乔海灵； 郭玉忠；

【作者基本信息】 郑州大学 ， 药理学， 2010， 硕士

【摘要】 研究目的本研究采用体外实验研究茵栀黄注射液及其主要活性成分对大鼠CYP2E1的抑制作用,并采用体内实验的方法研究茵栀黄注射液对CYP2E1的特异性底物氯畔沙宗的药动学的影响,在整体水平上评价茵栀黄注射液对大鼠CYP2E1的影响。研究方法1.茵栀黄注射液对CYP2E1的影响1.1茵栀黄注射液及其组分黄芩苷、栀子苷对大鼠CYP2E1的体外抑制作用1.1.1大鼠肝微粒体的制备取肝脏采用钙沉淀法制备大鼠肝微粒体。1.1.2微粒体蛋白的含量测定Bradfrod法测定微粒体蛋白含量。1.1.3孵育反应孵育体系总体积500μL,反应体系中包含NADPH,磷酸盐缓冲液,EDTA, MgCl2和微粒体蛋白。反应在37℃水浴中进行,预孵育5min,加入NADPH启动反应。实验分为对照组和实验组进行,实验组加入各成分,对照组加入等体积磷酸盐缓冲液。1.1.4酶活性的测定HPLC法测定氯唑沙宗的转化程度。1.2茵栀黄注射液连续给药对大鼠CYP2E1的抑制作用大鼠随机分为实验组和对照组,实验组每日静脉注射茵栀黄注射液(40m1·kg-1),对照组每日静脉注射相同剂量的葡萄糖注射液,连续给药7d,末次给药后即刻处死大鼠取肝脏制备肝微粒体,体外测定实验组和对照组大鼠氯唑沙宗的转化程度。1.3茵栀黄注射液对大鼠体内氯唑沙宗药动学的影响实验采用自身对照,14只雄性SD大鼠连续尾静脉注射10%葡萄糖注射液3d后,于末次给药后即刻,单次快速静脉注射氯唑沙宗注射液10mg·kg-1,并于2,8,20,40,70,100,150min采血0.5m1,离心分离血浆。首次采血3d后,14只大鼠尾静脉注射茵栀黄注射液(40m1·kg-1),连续给药7d,末次给药后静脉注射氯唑沙宗注射液10mg-kg-1,分别在给药后上述时间点采血0.5m1,离心分离血浆。HPLC法测定大鼠单用氯唑沙宗注射液组与合用茵栀黄注射液组氯唑沙宗的血药浓度。2.统计分析采用3P97药动学软件,计算氯唑沙宗药动学参数。应用SPSS10.0软件进行数据分析,实验组和对照组之间的比较采用独立样本t检验,氯唑沙宗单用组及与茵栀黄注射液合用组氯唑沙宗血药浓度及药动学参数比较采用配对t检验,P<0.05认为有统计学意义。结果1.茵栀黄注射液对CYP2E1的影响1.1茵栀黄注射液及其组分黄芩苷、栀子苷对大鼠CYP2E1的体外抑制作用茵栀黄注射液(P<0.001)、黄芩苷(P=0.002)、黄芩苷和栀子苷合用组(P=0.001)对CYP2E1有抑制作用,抑制率分别为66.79%,46.57%,52.46%；栀子苷(P=0.628)对CYP2E1无显著抑制作用。1.2茵栀黄注射液连续给药对大鼠CYP的2E1抑制作用连续静脉注射茵栀黄注射液7d后,实验组大鼠CYP2E1酶活性显著低于对照组(P<0.001),探针药物氯唑沙宗的代谢减少了约53.62%。1.3茵栀黄注射液对大鼠体内氯唑沙宗药动学的影响合用茵栀黄注射液后,单用组与合用组氯唑沙宗的主要药动学参数AUCo-t值分别为1202.31±157.34、926.93±169.7(μg·m1-1)·min,合用组较单用组有显著降低(P=0.009)；CL值分别为0.014±0.002、0.018±0.004 m1·kg-1·mmin-’,合用组较单用组有显著升高(P=0.016)；T1/2分别为19.98±3.74、29.74±14.98 min,合用组较单用组有显著延长(P=0.001)。结论1.茵栀黄注射液对大鼠离体肝CYP2E1有明显抑制作用,其作用与黄芩苷有关。2.茵栀黄注射液多次给药对大鼠肝CYP2E1有明显抑制作用。3.茵栀黄注射液显著抑制CYP2E1探针药物氯唑沙宗在大鼠体内的药动学。更多还原

【Abstract】 ObjectiveThe study was designed to study the active component of Yinzhihuang injection on the activity of CYP2E1 in rat liver microsome. We also studied the effect of Yinzhihuang injection on the pharmacokinetics of the CYP2E1 subtrate chlorzoxazone. The aim was to evaluate the effect of Yinzhihuang injection on the activity of CYP2E1 and guide reasonable clinical medication.Method1.The effect of Yinzhihuang injection on the acticity of CYP2E11.1 The inhibition of baicalin and geniposide on the acticity of CYP2E1 in rat microsome1.1.1 Preparation of liver microsome Liver microsome was prepared by using calcium precipitation approach.1.1.2 Determination of protein concentration Protein concentration was determinated by Bradfrod approach. 1.1.3 Incubation reaction The total volume of incubation system was 500μL, which contained NADPH, phosphate buffer, EDTA, MgCl2, and liver microsome. The reaction was preincubated for 5 min at 37℃, and was started since NADPH was added. The study was divided into control group and experiment group. The experiment group contained the active component Yinzhihuang of injection, the control group contained the same volume of phosphate buffer.1.1.4 Measurement of CYP2E1 enzyme activity HPLC method was used to determinate the turnover of midazolam.1.2 The inhibition of CYP2E1 activity after multiple administration of Yinzhihuang injection Male SD rats were randomly were divided into experiment group and control group. The experiment group were intravenously given Yinzhihuang injection(40ml·kg-1) and the control group were given glucose injection for 7 d. Immediately after the last treatment, rats were killed by decapitation to prepare the liver microsome, and then the turnover of chlorzoxazone of the two group were determinated.1.3 Effect of Yinzhihuang injection on the pharmacokinetics of chlorzoxazone The study was conducted by self-control design.14 male SD rats were given intravenously 10% glucose injection (40ml·kg-1), then chlorzoxazone at a dose of 10mg·kg-1 was infused via the tail vein to rats immediately after the last injection. A blood sample was collected at 2,8,20,40,70,100,150min the plasma was stored for analysis.3 days after the first blood collection, the 14 rats were given Yinzhihuang injection(40ml·kg-1) for 7 days. The blood sample was collected at designed time point.The concentration of chlorzoxazone was determinated by HPLC method.2. Statistical analysis The pharmacokinetic parameters of chlorzoxazone were calculated by software 3P97. The analyses were performed by SPSS 10.0 statistical software. The data of different groups was analyzed with independent t test. The differences of the concentration and pharmacokinetic parameters of chlorzoxazone single and its combination of Yinzhihuang injection were compared with paired t test. A value of P<0.05 was considered to be statistically significant.Result1. The effect of Yinzhihuang injection on the acticity of CYP2E11.1 The inhibition of baicalin and geniposide on the acticity of CYP2E1 in rat microsome Yinzhihuang injection(P<0.001), baicalin(P=0.002) and baicalin combine geniposide group(P=0.001) had inhibition on the activity of CYP2E1, the inhibition ratio was 66.79%,46.57%,52.46%, respectively; while geniposide (P=0. 628) did not.1.2 The inhibition of CYP2E1 activity after multiple administration of Yinzhihuang injection There were significant differences in the activities of CYP2E1 between control group and experiment group, the activities of CYP2E1 were inhibited obviously by administration Yinzhihuang injection(P<0.001). It decreased the metabolism of chlorzoxazone by 53.62%。1.3 Effect of Yinzhihuang injection on the pharmacokinetics of chlorzoxazoneThe main pharmacokinetic parameters of single administration of chlorzoxazone group and coadministration of Yinzhihuang injection group:the values of AUC0→t(P=0.009) were 1202.3±157.34、926.93±169.7 (μg·mr-1)-min, CL(P=0.016) were 0.014±0.002、0.018±0.004 ml-kg-1·-min-1, both of which had significance statistically, while the values of T1/2(P=0.001) were 19.98±3.7、29.74±14.98 min, which also had significance statistically. Conclusion1. Yinzhihuang injection can inhibit CYP2E1 activity in rat microsome in vitro, and baicalin plays an important part in it.2. Yinzhihuang injection inhibits the activity of CYP2E1 after multiple administration.3. Continuous administration of Yinzhihuang injection inhibits the pharmacokinetics of chlorzoxazone in rats.更多还原